Molecular characterization of the gene coding for major outer membrane protein OmpA from Enterobacter aerogenes

The ompA gene from Enterobacter aerogenes was subcloned into a low-copy-number plasmid vector and the resultant plasmid, pTU7En, used to study its expression in Escherichia coli K12. Although the gene was strongly expressed and large amounts of OmpA protein were present in the outer membrane its product was not functionally identical to the E. coli polypeptide. In particular, the E. aerogenes OmpA protein was unable to confer sensitivity to OmpA-specific phages of E. coli. When the primary structure of the protein was deduced from the nucleotide sequence of its gene it was found that three domains differed extensively from the corresponding regions of the E. coli protein. As two of these are known to be exposed on the cell surface we inferred that these alterations are responsible for differences in the biological activity of the two proteins.     

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P(K)/P(Cl)= approximately 3), whereas TolC is more selective to cations (P(K)/P(Cl)= approximately 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

Inhibitors of antibiotic efflux pump in resistant Enterobacter aerogenes strains

Enterobacter aerogenes, a nosocomial pathogen, is frequently exhibiting multidrug resistance mechanisms associated with a change in membrane permeability. In clinical isolates, active efflux plays a prominent role in antibiotic resistance. We report here the effect of three unrelated compounds that are able to restore a noticeable antibiotic susceptibility to resistant strains. The targeting of various parameters which contribute to the efficacy of the efflux mechanism, such as energy, flux selectivity, or functional assembly of the membrane complex, increases the intracellular chloramphenicol concentration in resistant isolates.     

Exopolysaccharide and storage polymer production in Enterobacter aerogenes type 8 strains

Many exopolysaccharide (EPS)-producing bacterial strains also synthesize storage polymers. The production of slime EPS and of the storage polymer glycogen was compared in batch cultures of EPS⁺ and EPS^- isogenic strains of Enterobacter aerogenes type 8. Conditions of nutrient imbalance with high C: N ratios favoured both EPS and storage polymer synthesis and resulted in little subsequent degradation of glycogen. In the EPS⁺ strain, glycogen synthesis was consistently lower than in the EPS^- strain, indicating that substrate was preferentially used for EPS production. Reduced levels of carbon substrate in the growth medium resulted in lower storage polymer synthesis and in the degradation of the glycogen formed in EPS-producing bacteria. Considerable differences in the synthesis and breakdown of intracellular carbohydrate were observed between bacteria grown in synthetic media with ammonium salts and the same bacteria grown in medium with casein hydrolysate as the nitrogen source. Growth in media depleted in magnesium was slower than in complete media but high yields of glycogen were obtained in both the EPS⁺ and EPS^- strains.     

Possible role of membrane proteins in mercury resistance of Enterobacter aerogenes

Mercury resistance shown by a strain of Enterobacter aerogenes was found to be determined by a plasmid. The resistance appeared to be not due to enzymatic volatilization of mercury, but due to the alteration in cellular permeability to mercury.Comparison of the outer membrane proteins was made between the resistant cells and the sensitive counterparts obtained by the treatment with mitomycin C, showing that two proteins with molecular weight of 46,000 and 44,000 had disappeared from the outer membrane along with the plasmid by the curing. These results suggest that the two membrane proteins mediating the cellular permeability to mercury compound may be responsible for the mercury resistance of the strain.     

Functional recruitment of human complement inhibitor C4B-binding protein to outer membrane protein Rck of Salmonella

Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH), we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP). Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs) 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.     

Cloning and expression in Escherichia coli K-12 of the genes for major outer membrane protein OmpA from Shigella dysenteriae, Enterobacter aerogenes, and Serratia marcescens.

The outer membranes of many gram-negative bacteria contain a major heat-modifiable protein which shows serological cross-reactivity with the OmpA protein of Escherichia coli K-12. Using the cloned gene for the E. coli K12 protein as a DNA-DNA hybridization probe, we were able to identify the corresponding genes from Shigella dysenteriae. Enterobacter aerogenes, and Serratia marcescens. These were cloned in a phage lambda vector, and their expression in E. coli K-12 was studied. All three OmpA proteins were fully produced and correctly exported to the outer membrane. In several cases, complete or partial restoration of known function of the E. coli K-12 protein was observed.     

Mutants defective in the 33K outer membrane protein of Salmonella typhimurium.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.     

Major outer membrane protein in Salmonella typhimurium induced by maltose.

A maltose-induced major outer membrane protein (the 44K protein) is demonstrated in Salmonella typhimurium. This protein resembles the lambda receptor of Escherichia coli in its location, induction properties, apparent molecular weight, and association with the peptidoglycan layer of the cell wall. The 44K protein is missing in certain Salmonella Mal- mutants, which are also missing a protein analogous to the maltose-binding protein of E. coli. Thus, these mutants may be defective in the control of maltose genese in Salmonella. The proteins appear to be closely related, as indicated by cross-reaction of the Salmonella protein with the antiserum raised against the lambda receptor; however, they are not identical, since the peptide patterns obtained after limited proteolysis are completely different. Bacteriophage lambda does not use the 44K protein as a receptor.     

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage lambda 1059, using the Escherichia coli ompC gene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Evaluation of recombinant outer membrane protein based vaccine against Salmonella Typhimurium in birds

Food-borne diseases caused by Salmonella enterica from poultry sources represent an important public health problem and no reliable control by vaccination has proved effective despite research. The aim of the present study was to evaluate the use of recombinant OmpC protein for immunization of birds to elucidate its protection against virulent Salmonella Typhimurium. The recombinant OmpC protein was prepared after cloning and expressing ompC gene and was characterized by SDS-PAGE and Western blot analyses. The protein preparations were tested as vaccine candidate in layer birds by comparing the immune response, protection and organ clearance against crude lysate and control. The biologically functional recombinant 43 kDa truncated OmpC protein proved to be a good immunogen which induced a significantly high humoral immune response than control. At the same time, it primed a stable cell-mediated immune response. A protective index (based on faecal shedding of organism) of rOmpC based preparations ranged between 50 and 75% as observed for 3 weeks after challenge. Therefore, the protein preparations conferred satisfactory protection against challenge infections with virulent strains of S. Typhimurium as evidenced by limited faecal shedding and minimal detection of Salmonella from edible tissues and eggs. These findings suggest the possibility to explore the use of S. enterica OMP protein for the production of novel vaccine.     

Cloning and expression of the Neisseria meningitidis 5C outer membrane protein

The 5C outer membrane protein, one of the N. meningitidis class 5 proteins, was preferably expressed in bacteria isolated from the nasopharynx and its role in adhering to the mucosal cells and invading them as well as the development of anti-5C antibodies in healthy carriers was demonstrated. Anti-5C monoclonal antibodies are bactericidal in the presence of the human complement. The immunodominant region of the 5C protein is highly conserved among the different strains of N. meningitidis, and the opc gene, which encodes the protein, does not seem to show antigenic variations. Here the isolation of the opc gene from the Cuban strain B:4:P1.15 by PCR (Polymerase Chain Reaction) is presented. Under the regulation of the tryptophan promoter, the gene was cloned and sequenced in E. coli with a high level of expression and fused to the amino-terminal end of the interleukin-2 gene. In the dot-blot experiments, the presence of the gene in those strains which did not express the protein in the whole cell ELISA was also detectable.     

The outer membrane form of the mitochondrial protein Mcr1 follows a TOM-independent membrane insertion pathway

The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle.